西安建筑科技大学：《水资源利用与保护》科研项目及成果_PCR病原细菌检测项目

Rapid detection of pathogenic bacteria in surface water by bacteria universal primer Introduction The increase of urban population often results in higher percentage of untreated sewage into surface waters, and this is probably responsible for the great number of human morbidities and mortalities worldwide. In particular, waterbome infections such as typhoid fever, cholera, dysentery and traveler's diarrhea are caused by different types of bacterial pathogens and thus pose a major public health hazard, especially in developing countries. Regular monitoring of waterborne pathogens is extremely important for the protection of public health. The present study is a preliminary work to establish a rapid detection method using PCR with universal primers. The target bacteria are the common pathogenic bacteria in surface water which are related to the most frequently occurred waterborne deceases Results Discussion PCR amplification and analysis of reference strains PCR analyses and bacteria enumeration of surface The universal primer pair was tested by PCr on DNA templates prepared water samples from Shigella dysenteriae, Vibrio cholerae, Salmonella typhimurium, and Using the universal primer PCR protocol mentioned above, PCR analyses Escherichia coli. As shown in Fig. 1, a limpid specific strap could e seen were conducted for water samples collected from the 5 location. For at 320bp for all the four strains on the electrophoregrams. Analysis results comparison, bacteria counting was also conducted regarding total bacteria verified that the universal primer pair showed specificities only for the and faecal coliform. For PCR analyses, bacterial cells were harvested corresponding target genes from the each of the water samples by centrifugation and then total DNA was recovered by phenol-chloroform extraction. As shown in Fig 3 among the 5 water samples, PCR products were detected from 4 samples and their band intensities were in a sequence of No 4> No. 3> No. 1> No 5. Regarding sample No. 2 which was collected from Heihe River, a well protected water source for potable water supply, there were no any detectable PCR products. Fig 4 shows the bacteria enumeration results The numbers of total bacteria in the 5 water samples were in a sequence of No 4 >No. 1> No 5> No. 3> No. 1. Regarding faecal coliform, it was Fig 1. PCR amplification results of the four reference strains detected only from 2 water samples, namely No, 4 and No. 3 M: DNA marker, 1: Shigella dysenteriae, 2: Vibrio cholerae, 3: Salmonella typhimurium, 4: Escherichia coli, 5: Negative control Determination of detection sensitivity of the universal primer PCR protocol The detection sensitivity of the universal primer PCR for the Escherichia coli was examined in serial 10-fold dilutions in autoclaved distilled water Cell suspensions containing 2.75X 100 cfu/100mL-275 X 106 cfu/100mL Fig 3. PCR detection results of surface water sample. ck: negative control of Escherichia coli. reference strain was harvested by centrifugation. The 1: Chanhe River, 2: Heihe River, 3: Xingqinghu Lake, 4: Secondary effluent otal DNA from bacteria at each serial dilution was extracted by phenol- (undisinfected), 5: Beihu Lake, ckt: positive control, (E coli. 100cfu/100mL). chloroform and tested with the universal primer PCR. As shown in Fig. 2, the detection limit of E. coli was evaluated to be 27.5 cfu/100mL(Fig. 2a line 6), and the detection limit of the chromosome DNA for E. coli was evaluated to be 250 ng/L (Figure 2b, line 5). Similar results were obtained in at least four independent experiments Kb M Fig. 4 Enumeration of total bacteria and faecal coliform in surface 0.7 Sample No 4 is the undisinfected secondary effluent from a domestic wastewater treatment plant. As anticipated, the PCR band intensity of this sample was very strong and the numbers of total bacteria and faecal coliform were both very high(about 20000 cfu/mL and 70 cfu/100mL, respectively). Sample No. 3 from which the PCR band intensity was strong Fig 2. The PCR sensibility detection of the universal primer and higher number of faecal coliform was detected(about 55 cfu/100mL) (a) PCR amplification results of the E coli suspension under serially 10-fold is the water from the Xingqing Lake where water pollution used to be very diluted in sterile distilled water. M: Marker. 1: 2.75X 106 cfu/100mL serious before due to intrusion of urban drainage and domestic wastes 2:275×105cfu/100mL,3:2.75×104cfu/100mL,4:275×103 and water quality improvement has just been implemented by sediment cfu/100mL,5:275×102cfu/100mL,6:2.75×101cfu100mL,7 dredging and pollution source control. The result indicated that the water 2.75X 100 cfu/100mL, 8: Negative control quality is still bacteriologically unfavorable Regarding sample No. 1 which (b) PCR amplification results of the total DNA of E coli. under serially 10- is from a river receiving urban drainage and certain industrial wastewater, fold diluted in sterile distilled water. M: Marker, 1: 2.5mg/L, 2: 250ug/L, although no faecal coliform was detected the total bacteria count was as 3: 25u g/L, 4: 2.5ug/L, 5: 250ng/L, 6: 25ng/L, 7: Negative control high as 16000 cfu/mL and PCR products were also detected Conclusions With its rapidness and sensibility for the detection of pathogenic bacteria from surface water, the universal primer PCR method established by this study can provide a more reliable approach for bacteriological analysis of water environment from the viewpoint of safeguard of public health Acknowledgement: This study is supported by the National Natural Science Foundation of China(Grant No. 50478048)

Rapid detection of pathogenic bacteria in surface water by bacteria universal primer The increase of urban population often results in higher percentage of untreated sewage into surface waters, and this is probably responsible for the great number of human morbidities and mortalities worldwide. In particular, waterborne infections such as typhoid fever, cholera, dysentery and traveler’s diarrhea are caused by different types of bacterial pathogens and thus pose a major public health hazard, especially in developing countries. Regular monitoring of waterborne pathogens is extremely important for the protection of public health.The present study is a preliminary work to establish a rapid detection method using PCR with universal primers. The target bacteria are the common pathogenic bacteria in surface water which are related to the most frequently occurred waterborne deceases. Introduction Results & Discussion The detection sensitivity of the universal primer PCR for the Escherichia coli. was examined in serial 10-fold dilutions in autoclaved distilled water. Cell suspensions containing 2.75×100 cfu/100mL–2.75×106 cfu/100mL of Escherichia coli. reference strain was harvested by centrifugation. The total DNA from bacteria at each serial dilution was extracted by phenol– chloroform and tested with the universal primer PCR. As shown in Fig. 2, the detection limit of E. coli. was evaluated to be 27.5 cfu/100mL (Fig. 2a, line 6), and the detection limit of the chromosome DNA for E. coli. was evaluated to be 250 ng/L (Figure 2b, line 5). Similar results were obtained in at least four independent experiments. Conclusions The universal primer pair was tested by PCR on DNA templates prepared from Shigella dysenteriae, Vibrio cholerae, Salmonella typhrmurium, and Escherichia coli. As shown in Fig. 1, a limpid specific strap could be seen at 320bp for all the four strains on the electrophoregrams. Analysis results verified that the universal primer pair showed specificities only for their corresponding target genes. Fig 2. The PCR sensibility detection of the universal primer. (a) PCR amplification results of the E.coli suspension under serially 10-fold diluted in sterile distilled water. M: Marker, 1: 2.75×106 cfu/100mL, 2: 2.75×105 cfu/100mL, 3: 2.75×104 cfu/100mL, 4: 2.75×103 cfu/100mL, 5: 2.75×102 cfu/100mL, 6: 2.75×101 cfu/100mL, 7: 2.75×100 cfu/100mL, 8: Negative control. (b) PCR amplification results of the total DNA of E.coli. under serially 10- fold diluted in sterile distilled water. M: Marker, 1: 2.5mg/L, 2: 250μg/L, 3: 25μ g/L, 4: 2.5μg/L, 5: 250ng/L, 6: 25ng/L, 7: Negative control. With its rapidness and sensibility for the detection of pathogenic bacteria from surface water, the universal primer PCR method established by this study can provide a more reliable approach for bacteriological analysis of water environment from the viewpoint of safeguard of public health. Fig 1. PCR amplification results of the four reference strains M: DNA marker, 1: Shigella dysenteriae, 2: Vibrio cholerae, 3: Salmonella typhrmurium, 4: Escherichia coli., 5: Negative control PCR amplification and analysis of reference strains Determination of detection sensitivity of the universal primer PCR protocol PCR analyses and bacteria enumeration of surface water samples Using the universal primer PCR protocol mentioned above, PCR analyses were conducted for water samples collected from the 5 location. For comparison, bacteria counting was also conducted regarding total bacteria and faecal coliform. For PCR analyses, bacterial cells were harvested from the each of the water samples by centrifugation and then total DNA was recovered by phenol–chloroform extraction. As shown in Fig.3, among the 5 water samples, PCR products were detected from 4 samples and their band intensities were in a sequence of No.4 > No.3 > No.1 > No.5. Regarding sample No. 2 which was collected from Heihe River, a well protected water source for potable water supply, there were no any detectable PCR products. Fig. 4 shows the bacteria enumeration results. The numbers of total bacteria in the 5 water samples were in a sequence of No.4 > No.1 > No.5 > No.3 > No.1. Regarding faecal coliform, it was detected only from 2 water samples, namely No.4 and No.3. (a) (b) Fig 3. PCR detection results of surface water sample.ck – : negative control, 1: Chanhe River, 2: Heihe River, 3: Xingqinghu Lake, 4: Secondary effluent (undisinfected), 5: Beihu Lake, ck + : positive control,(E. coli. 100cfu/100mL). 0 5000 10000 15000 20000 25000 1 2 3 4 5 Total bacterium (cfu/mL) mL) 0 2 0 4 0 6 0 8 0 100 Faecal coliform (cfu/100mL) mL) 1 2 3 4 5 Fig. 4 Enumeration of total bacteria and faecal coliform in surface water samples. Sample No.4 is the undisinfected secondary effluent from a domestic wastewater treatment plant. As anticipated, the PCR band intensity of this sample was very strong and the numbers of total bacteria and faecal coliform were both very high (about 20000 cfu/mL and 70 cfu/100mL, respectively). Sample No.3 from which the PCR band intensity was strong and higher number of faecal coliform was detected (about 55 cfu/100mL) is the water from the Xingqing Lake where water pollution used to be very serious before due to intrusion of urban drainage and domestic wastes and water quality improvement has just been implemented by sediment dredging and pollution source control. The result indicated that the water quality is still bacteriologicaly unfavorable. Regarding sample No.1 which is from a river receiving urban drainage and certain industrial wastewater, although no faecal coliform was detected, the total bacteria count was as high as 16000 cfu/mL and PCR products were also detected. Acknowledgement: This study is supported by the National Natural Science Foundation of China (Grant No. 50478048)